Cocaine addiction is a chronic relapsing disorder characterized by compulsive cocaine use. Over the past few decades, NIDA has very actively supported clinical and preclinical research designed to understand the motivational and neurobiological impacts of factors capable of promoting cocaine seeking and craving. Unfortunately, most of the interventions (e.g., anti-craving medications) aimed to block these relapse- promoting factors have been proven to be ineffective in clinical trials. Therefore, we believe that it is critical to make a strategic shift to investigate the factors capable of suppresing (as opposed to promoting) cocaine seeking, craving and ultimately relapse. On this premise, we developed an omission cue-induced suppression (OCIS) paradigm in order to systematically evaluate the relapse-suppressing potential of cues signaling cocaine omission (unavailability) in rats with a cocaine history associated with compulsive cocaine use. The preliminary results demonstrate for the first time that omission cues are capable of suppressing addiction-like cocaine seeking. The fact that OCIS and extinction of cocaine seeking significantly differ at behavioral level (unlike OCIS, extinction is ineffective at suppressing the relapse-promoting capacity of cocaine availability cues and cocaine itself) leaves open the possibility that they als differ at neurobiological level. Using the OCIS paradigm as the base for the proposed experiments, this project will test the overarching hypothesis that the OCIS of addiction-like cocaine-seeking habit involves distinct neuronal ensembles within the reward circuit. In Specific Aim 1, Fos/NeuN double-label staining (an immunohistochemical tool to map activated neuronal ensembles) will be used to localize neuronal ensembles activated by omission cues. In Specific Aim 2, the Daun02 inactivation method (a novel pharmacogenetic technique to selectively disrupt Fos positive activated neurons) will be used to determine the neuronal ensembles within the medial prefrontal cortex mediating OCIS. In line with the scope of R21 applications, the proposed experiments are exploratory and developmental but possibly with high reward for several reasons. First, no study has yet systematically examined the brain mechanisms that suppress cocaine seeking in rats with a drug history associated with compulsive cocaine use. Second, the Daun02 inactivation method has not been applied to determine neuronal ensembles mediating any form of drug-seeking suppression. Importantly, this is the only method currently available to examine the behavioral contribution of specific neuronal ensembles. Third, nothing is known of the neurobiology of OCIS. In summary, the proposed project is designed to provide the groundwork for future systemic research to uncover the brain mechanisms underlying the robust relapse-suppressing potential of omission cues. Such investigation may lead to more effective interventions to treat the chronically relapsing nature of cocaine addiction.